Regulation of the apico-basolateral trafficking polarity of the homologous copper-ATPases ATP7A and ATP7B.

The homologous P-type copper-ATPases (Cu-ATPases) ATP7A and ATP7B are the key regulators of copper homeostasis in mammalian cells. In polarized epithelia, upon copper treatment, ATP7A and ATP7B traffic from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively. We characterized the sorting pathways of Cu-ATPases between TGN and the plasma membrane and identified the machinery involved. ATP7A and ATP7B reside on distinct domains of TGN in limiting copper conditions, and in high copper, ATP7A traffics to basolateral membrane, whereas ATP7B traverses common recycling, apical sorting and apical recycling endosomes en route to apical membrane. Mass spectrometry identified regulatory partners of ATP7A and ATP7B that include the adaptor protein-1 complex. Upon knocking out pan-AP-1, sorting of both Cu-ATPases is disrupted. ATP7A loses its trafficking polarity and localizes on both apical and basolateral surfaces in high copper. By contrast, ATP7B loses TGN retention but retained its trafficking polarity to the apical domain, which became copper independent. Using isoform-specific knockouts, we found that the AP-1A complex provides directionality and TGN retention for both Cu-ATPases, whereas the AP-1B complex governs copper-independent trafficking of ATP7B solely. Trafficking phenotypes of Wilson disease-causing ATP7B mutants that disrupts putative ATP7B-AP1 interaction further substantiates the role of AP-1 in apical sorting of ATP7B.

Organization and execution of the epithelial polarity programme.

Epithelial cells require apical-basal plasma membrane polarity to carry out crucial vectorial transport functions and cytoplasmic polarity to generate different cell progenies for tissue morphogenesis. The establishment and maintenance of a polarized epithelial cell with apical, basolateral and ciliary surface domains is guided by an epithelial polarity programme (EPP) that is controlled by a network of protein and lipid regulators. The EPP is organized in response to extracellular cues and is executed through the establishment of an apical-basal axis, intercellular junctions, epithelial-specific cytoskeletal rearrangements and a polarized trafficking machinery. Recent studies have provided insight into the interactions of the EPP with the polarized trafficking machinery and how these regulate epithelial polarization and depolarization.

Lipofuscin causes atypical necroptosis through lysosomal membrane permeabilization.

Lipofuscin granules enclose mixtures of cross-linked proteins and lipids in proportions that depend on the tissue analyzed. Retinal lipofuscin is unique in that it contains mostly lipids with very little proteins. However, retinal lipofuscin also presents biological and physicochemical characteristics indistinguishable from conventional granules, including indigestibility, tendency to cause lysosome swelling that results in rupture or defective functions, and ability to trigger NLRP3 inflammation, a symptom of low-level disruption of lysosomes. In addition, like conventional lipofuscins, it appears as an autofluorescent pigment, considered toxic waste, and a biomarker of aging. Ocular lipofuscin accumulates in the retinal pigment epithelium (RPE), whereby it interferes with the support of the neuroretina. RPE cell death is the primary cause of blindness in the most prevalent incurable genetic and age-related human disorders, Stargardt disease and age-related macular degeneration (AMD), respectively. Although retinal lipofuscin is directly linked to the cell death of the RPE in Stargardt, the extent to which it contributes to AMD is a matter of debate. Nonetheless, the number of AMD clinical trials that target lipofuscin formation speaks for the potential relevance for AMD as well. Here, we show that retinal lipofuscin triggers an atypical necroptotic cascade, amenable to pharmacological intervention. This pathway is distinct from canonic necroptosis and is instead dependent on the destabilization of lysosomes. We also provide evidence that necroptosis is activated in aged human retinas with AMD. Overall, this cytotoxicity mechanism may offer therapeutic targets and markers for genetic and age-related diseases associated with lipofuscin buildups.

Copper(II) import and reduction are dependent on His-Met clusters in the extracellular amino terminus of human copper transporter-1.

Copper(I) is an essential metal for all life forms. Though Cu(II) is the most abundant and stable state, its reduction to Cu(I) via an unclear mechanism is prerequisite for its bioutilization. In eukaryotes, the copper transporter-1 (CTR1) is the primary high-affinity copper importer, although its mechanism and role in Cu(II) reduction remain uncharacterized. Here we show that extracellular amino-terminus of human CTR1 contains two methionine-histidine clusters and neighboring aspartates that distinctly bind Cu(I) and Cu(II) preceding its import. We determined that hCTR1 localizes at the basolateral membrane of polarized MDCK-II cells and that its endocytosis to Common-Recycling-Endosomes is regulated by reduction of Cu(II) to Cu(I) and subsequent Cu(I) coordination by the methionine cluster. We demonstrate the transient binding of both Cu(II) and Cu(I) during the reduction process is facilitated by aspartates that also act as another crucial determinant of hCTR1 endocytosis. Mutating the first Methionine cluster (7Met-Gly-Met9) and Asp13 abrogated copper uptake and endocytosis upon copper treatment. This phenotype could be reverted by treating the cells with reduced and nonreoxidizable Cu(I). We show that histidine clusters, on other hand, bind Cu(II) and are crucial for hCTR1 functioning at limiting copper. Finally, we show that two N-terminal His-Met-Asp clusters exhibit functional complementarity, as the second cluster is sufficient to preserve copper-induced CTR1 endocytosis upon complete deletion of the first cluster. We propose a novel and detailed mechanism by which the two His-Met-Asp residues of hCTR1 amino-terminus not only bind copper, but also maintain its reduced state, crucial for intracellular uptake.

Accurate measurement of fast endocytic recycling kinetics in real time.

The fast turnover of membrane components through endocytosis and recycling allows precise control of the composition of the plasma membrane. Endocytic recycling can be rapid, with some molecules returning to the plasma membrane with a half time <5 min. Existing methods to study these trafficking pathways utilize chemical, radioactive or fluorescent labeling of cell surface receptors in pulse-chase experiments, which require tedious washing steps and manual collection of samples. Here, we introduce a live-cell endocytic recycling assay based on a newly designed cell-impermeable fluorogenic ligand for HaloTag, Janelia Fluor 635i (JF635i, where i indicates impermeant), which allows real-time detection of membrane receptor recycling at steady state. We used this method to study the effect of iron depletion on transferrin receptor (TfR) recycling using the chelator desferrioxamine. We found that this perturbation significantly increases the TfR recycling rate. The high temporal resolution and simplicity of this assay provides a clear advantage over extant methods and makes it ideal for large scale cellular imaging studies. This assay can be adapted to examine other cellular kinetic parameters such as protein turnover and biosynthetic trafficking.

Apical CLC-2 in retinal pigment epithelium is crucial for survival of the outer retina.

Knockout of the chloride channel protein 2 (CLC-2; CLCN2) results in fast progressing blindness in mice. Retinal Pigment Epithelium (RPE) and photoreceptors undergo, in parallel, rapid, and profound morphological changes and degeneration. Immunohistochemistry and electron microscopy of the outer retina and electroretinography of the CLC-2 KO mouse demonstrated normal morphology at postnatal day 2, followed by drastic changes in RPE and photoreceptor morphology and loss of vision during the first postnatal month. To investigate whether the RPE or the photoreceptors are the primary cause of the degeneration, we injected lentiviruses carrying HA-tagged CLC-2 with an RPE-specific promotor in the subretinal space of CLC-2-KO mice at the time of eye opening. As expected, CLC-2-HA was expressed exclusively in RPE; strikingly, this procedure rescued the degeneration of both RPE and photoreceptors. Light response in transduced eyes was also recovered. Only a fraction of RPE was transduced with the lentivirus; however, the entire RPE monolayer appears healthy, even the RPE cells not expressing the CLC-2-HA. Surprisingly, in contrast with previous physiological observations that postulate that CLC-2 has a basolateral localization in RPE, our immunofluorescence experiments demonstrated CLC-2 has an apical distribution, facing the subretinal space and the photoreceptor outer segments. Our findings suggest that CLC-2 does not play the postulated role in fluid transport at the basolateral membrane. Rather, they suggest that CLC-2 performs a critical homeostatic role in the subretinal compartment involving a chloride regulatory mechanism that is critical for the survival of both RPE and photoreceptors.

Quantitative proteomics of MDCK cells identify unrecognized roles of clathrin adaptor AP-1 in polarized distribution of surface proteins.

The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity was observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now.

The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia.

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis.

IQGAP1 controls tight junction formation through differential regulation of claudin recruitment.

IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42-JNK pathway.

Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates.

Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy. Our experiments directly demonstrate that rhodopsin-GFP traffics through apical recycling endosomes (AREs) that bear the small GTPase Rab11a before arriving at the apical membrane. Expression of dominant-negative Rab11a drastically reduced apical delivery of rhodopsin-GFP and caused its missorting to the basolateral membrane. Surprisingly, functional inhibition of dynamin-2 trapped rhodopsin-GFP at AREs and caused aberrant accumulation of coated vesicles on AREs, suggesting a previously unrecognized role for dynamin-2 in the scission of apical carrier vesicles from AREs. A second set of experiments, using a unique method to carry out total internal reflection fluorescence microscopy (TIRFM) from the apical side, allowed us to visualize the fusion of rhodopsin-GFP carrier vesicles, which occurred randomly all over the apical plasma membrane. Furthermore, two-color TIRFM showed that Rab11a-mCherry was present in rhodopsin-GFP carrier vesicles and was rapidly released upon fusion onset. Our results provide direct evidence for a role of AREs as a post-Golgi sorting hub in the biosynthetic route of polarized epithelia, with Rab11a regulating cargo sorting at AREs and carrier vesicle docking at the apical membrane.

Beta cyclodextrins bind, stabilize, and remove lipofuscin bisretinoids from retinal pigment epithelium.

Accumulation of lipofuscin bisretinoids (LBs) in the retinal pigment epithelium (RPE) is the alleged cause of retinal degeneration in genetic blinding diseases (e.g., Stargardt) and a possible etiological agent for age-related macular degeneration. Currently, there are no approved treatments for these diseases; hence, agents that efficiently remove LBs from RPE would be valuable therapeutic candidates. Here, we show that beta cyclodextrins (ß-CDs) bind LBs and protect them against oxidation. Computer modeling and biochemical data are consistent with the encapsulation of the retinoid arms of LBs within the hydrophobic cavity of ß-CD. Importantly, ß-CD treatment reduced by 73% and 48% the LB content of RPE cell cultures and of eyecups obtained from Abca4-Rdh8 double knock-out (DKO) mice, respectively. Furthermore, intravitreal administration of ß-CDs reduced significantly the content of bisretinoids in the RPE of DKO animals. Thus, our results demonstrate the effectiveness of ß-CDs to complex and remove LB deposits from RPE cells and provide crucial data to develop novel prophylactic approaches for retinal disorders elicited by LBs.

Galectin-4-mediated transcytosis of transferrin receptor.

Some native epithelia, for example, retinal pigment epithelium (RPE) and kidney proximal tubule (KPT), constitutively lack the basolateral sorting adaptor AP-1B; this results in many basolateral plasma membrane proteins being repositioned to the apical domain, where they perform essential functions for their host organs. We recently reported the underlying apical polarity reversal mechanism: in the absence of AP-1B-mediated basolateral sorting, basolateral proteins are shuttled to the apical plasma membrane through a transcytotic pathway mediated by the plus-end kinesin KIF16B. Here, we demonstrate that this apical transcytotic pathway requires apical sorting of basolateral proteins, which is mediated by apical signals and galectin-4. Using RPE and KPT cell lines, and AP-1B-knockdown MDCK cells, we show that mutation of the N-glycan linked to N727 in the basolateral marker transferrin receptor (TfR) or knockdown of galectin-4 inhibits TfR transcytosis to apical recycling endosomes and the apical plasma membrane, and promotes TfR lysosomal targeting and subsequent degradation. Our results report a new role of galectins in basolateral to apical epithelial transcytosis.

Conditional knockdown of DNA methyltransferase 1 reveals a key role of retinal pigment epithelium integrity in photoreceptor outer segment morphogenesis.

Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.

Basolateral sorting of the coxsackie and adenovirus receptor through interaction of a canonical YXXPhi motif with the clathrin adaptors AP-1A and AP-1B.

The coxsackie and adenovirus receptor (CAR) plays key roles in epithelial barrier function at the tight junction, a localization guided in part by a tyrosine-based basolateral sorting signal, (318)YNQV(321). Sorting motifs of this type are known to route surface receptors into clathrin-mediated endocytosis through interaction with the medium subunit (µ2) of the clathrin adaptor AP-2, but how they guide new and recycling membrane proteins basolaterally is unknown. Here, we show that YNQV functions as a canonical YxxΦ motif, with both Y318 and V321 required for the correct basolateral localization and biosynthetic sorting of CAR, and for interaction with a highly conserved pocket in the medium subunits (µ1A and µ1B) of the clathrin adaptors AP-1A and AP-1B. Knock-down experiments demonstrate that AP-1A plays a role in the biosynthetic sorting of CAR, complementary to the role of AP-1B in basolateral recycling of this receptor. Our study illustrates how two clathrin adaptors direct basolateral trafficking of a plasma membrane protein through interaction with a canonical YxxΦ motif.

A molecular rheostat at the interface of cancer and diabetes.

Epidemiology studies revealed the connection between several types of cancer and type 2 diabetes (T2D) and suggested that T2D is both a symptom and a risk factor of pancreatic cancer. High level of circulating insulin (hyperinsulinemia) in obesity has been implicated in promoting aggressive types of cancers. Insulin resistance, a symptom of T2D, pressures pancreatic ß-cells to increase insulin secretion, leading to hyperinsulinemia, which in turn leads to a gradual loss of functional ß-cell mass, thus indicating a fine balance and interplay between ß-cell function and mass. While the mechanisms of these connections are unclear, the mTORC1-Akt signaling pathway has been implicated in controlling ß-cell function and mass, and in mediating the link of cancer and T2D. However, incomplete understating of how the pathway is regulated and how it integrates body metabolism has hindered its efficacy as a clinical target. The IQ motif containing GTPase activating protein 1 (IQGAP1)-Exocyst axis is a growth factor- and nutrient-sensor that couples cell growth and division. Here we discuss how IQGAP1-Exocyst, through differential interactions with Rho-type of small guanosine triphosphatases (GTPases), acts as a rheostat that modulates the mTORC1-Akt and MAPK signals, and integrates ß-cell function and mass with insulin signaling, thus providing a molecular mechanism for cancer initiation in diabetes. Delineating this regulatory pathway may have the potential of contributing to optimizing the efficacy and selectivity of future therapies for cancer and diabetes.

Mechanism of polarized lysosome exocytosis in epithelial cells.

Fusion of lysosomes with the plasma membrane is a calcium-dependent process that is crucial for membrane repair, limiting pathogen entry and clearing cellular debris. In non-polarized cells, lysosome exocytosis facilitates rapid resealing of torn membranes. Here, we investigate the mechanism of lysosome exocytosis in polarized epithelia, the main barrier between the organism and the external environment and the first line of defense against pathogens. We find that in polarized Madin-Darby canine kidney (MDCK) cells, calcium ionophores or pore-forming toxins cause lysosomes to fuse predominantly with the basolateral membrane. This polarized exocytosis is regulated by the actin cytoskeleton, membrane cholesterol and the clathrin adaptor AP-1. Depolymerization of actin, but not microtubules, causes apical lysosome fusion, supporting the hypothesis that cortical actin is a barrier to exocytosis. Overloading lysosomes with cholesterol inhibits exocytosis, suggesting that excess cholesterol paralyzes lysosomal traffic. The clathrin adaptor AP-1 is responsible for accurately targeting syntaxin 4 to the basolateral domain. In cells lacking either the ubiquitous AP-1A or the epithelial-specific AP-1B, syntaxin 4 is non-polar. This causes lysosomes to fuse with both the apical and basolateral membranes. Consistent with these findings, RNAi-mediated depletion of syntaxin 4 inhibits basolateral exocytosis in wild-type MDCK, and both apical and basolateral exocytosis in cells lacking AP-1A or AP-1B. Our results provide fundamental insight into the molecular machinery involved in membrane repair in polarized epithelia and suggest that AP-1 is a crucial regulator of this process.

Plasma membrane protein polarity and trafficking in RPE cells: past, present and future.

The retinal pigment epithelium (RPE) comprises a monolayer of polarized pigmented epithelial cells that is strategically interposed between the neural retina and the fenestrated choroid capillaries. The RPE performs a variety of vectorial transport functions (water, ions, metabolites, nutrients and waste products) that regulate the composition of the subretinal space and support the functions of photoreceptors (PRs) and other cells in the neural retina. To this end, RPE cells display a polarized distribution of channels, transporters and receptors in their plasma membrane (PM) that is remarkably different from that found in conventional extra-ocular epithelia, e.g. intestine, kidney, and gall bladder. This characteristic PM protein polarity of RPE cells depends on the interplay of sorting signals in the RPE PM proteins and sorting mechanisms and biosynthetic/recycling trafficking routes in the RPE cell. Although considerable progress has been made in our understanding of the RPE trafficking machinery, most available data have been obtained from immortalized RPE cell lines that only partially maintain the RPE phenotype and by extrapolation of data obtained in the prototype Madin-Darby Canine Kidney (MDCK) cell line. The increasing availability of RPE cell cultures that more closely resemble the RPE in vivo together with the advent of advanced live imaging microscopy techniques provides a platform and an opportunity to rapidly expand our understanding of how polarized protein trafficking contributes to RPE PM polarity.

Microfluidic approaches for epithelial cell layer culture and characterisation.

In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips; including methods to perform electrical impedance spectroscopy; methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry; techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress; and methods to carry out high-resolution imaging of vesicular trafficking using light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers.

Clathrin is a key regulator of basolateral polarity.

Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.

Basolateral sorting signals regulating tissue-specific polarity of heteromeric monocarboxylate transporters in epithelia.

Many solute transporters are heterodimers composed of non-glycosylated catalytic and glycosylated accessory subunits. These transporters are specifically polarized to the apical or basolateral membranes of epithelia, but this polarity may vary to fulfill tissue-specific functions. To date, the mechanisms regulating the tissue-specific polarity of heteromeric transporters remain largely unknown. Here, we investigated the sorting signals that determine the polarity of three members of the proton-coupled monocarboxylate transporter (MCT) family, MCT1, MCT3 and MCT4, and their accessory subunit CD147. We show that MCT3 and MCT4 harbor strong redundant basolateral sorting signals (BLSS) in their C-terminal cytoplasmic tails that can direct fusion proteins with the apical marker p75 to the basolateral membrane. In contrast, MCT1 lacks a BLSS and its polarity is dictated by CD147, which contains a weak BLSS that can direct Tac, but not p75 to the basolateral membrane. Knockdown experiments in MDCK cells indicated that basolateral sorting of MCTs was clathrin-dependent but clathrin adaptor AP1B-independent. Our results explain the consistently basolateral localization of MCT3 and MCT4 and the variable localization of MCT1 in different epithelia. They introduce a new paradigm for the sorting of heterodimeric transporters in which a hierarchy of apical and BLSS in the catalytic and/or accessory subunits regulates their tissue-specific polarity.